(A) Technical Field of the Invention
The present invention provides a method of separating casein from animal milk by means of ceramic filtering membrane, and its subsequent applications.
(B) Description of the Prior Art
Human protein medicines such as Insulin, Erythropoietin (EPO), and Human Growth Hormone (HGH), etc. have been put to use in clinical practice for years with fairly good medicinal effects; however, because in the past the sources of such medicines could only be acquired from natural blood, tissues, or organs, the production volume was very low and the prices were high, making it difficult for the ordinary people to afford them. In recent years, with the advancement of biological technologies, human beings have been able to mass-produce these types of medicines by using the technology of recombinant DNA, and, as the result, the production costs of human protein medicines have been lowered significantly.
Among various systems of producing recombinant protein medicines, transgenic animals, because of its being able to produce within their bodies a large amount of desired target protein, are found to be of more potentiality in being commercialized than yeast or zooblast. By transferring the foreign genes inside the bodies of mammals, a large amount of recombinant target protein can be purified from the milk they excrete; however, because milk is a sophisticated bio-colloid, the complexity of its composition is equivalent to that of plasma, hence causing difficulty in the process of purification.
Generally speaking, the composition of the endogenous protein in the milk of transgenic animals does not differ from that of non transgenic animals simply because it contains recombinant target protein. Therefore, the amount of recombinant target protein present in the milk of transgenic animals will affect the amount of difficulty involved in the purification; theoretically, the more the present amount of foreign gene, the better. But for some complicated structured proteins, due to the concerns over their modifications, the amount present is usually not set very high.
The proteins in the milk are mainly composed of casein, and the content of casein is over 50% of the total amount of protein; therefore, casein often becomes the source of impurity in the purification process of the protein. Casein exists in milk in the form of micelles. Take the milk of pigs as an example. Casein is bonded with colloidal calcium phosphate to form in the milk suspending particles of 133 nm in diameter on the average. Furthermore, plasmin is also a component packed full of in milk which is able to hydrolyze the peptide bonds between Lys-X and Arg-X, while the optimal parameter for its activity in hydrolysis is pH value 7.5, at 37° C. Hence, after the milk sample is collected, the key concern will be on how to avoid the effect by the plasmin. Since plasmin exists in milk by means of bonding with the micelles of casein, the separation of recombinant protein from milk should first consider the removing the casein micelles; however, it is rather a difficult and an annoying process in the situations of practical operation.
The existing methods of removing the casein micelles include the first method by adjusting the pH of the milk to 4.6 or below to allow the milk separated into precipitate of acid based milk curds and whey that contains soluble recombinant protein, but the drawback of this method is that the change of pH value often destroy the structure of protein, depriving of its activity; besides, if the recombinant protein itself is an acidic protein, it will precipitate at such pH value and not able to be separated; furthermore, the condition of acidity will cause the residual base of sialic acid to be removed from carbohydrate protein, resulting in the change of composition to the protein. The second method uses PEG to enable the casein micelles to precipitate, but the prerequisite is that the recombinant target protein cannot be precipitated. Apart from the restrictions aforesaid for these two methods, it takes the centrifugal force of a super high performance centrifuge to make normal casein micelles precipitate when no treatment is applied; such a requirement of high performance centrifugal force for separating the precipitates and the clear fluid on top actually causes lots of inconvenience in operation high volume. The third method is using EDTA or citrate to dissolve the casein micelles, and then process through ultrafiltration. Ultrafiltration is a process commonly used in the manufacturing of organic medicinal preparations (such as medical protein, serum, antibody, etc.), the main function of which is to remove the microorganism or virus in the biologicals, or to remove small molecules of salts in solutions. As milk is a sophisticated type of bio-colloid, its micelles tend to block the filtering membrane during ultrafiltration, posing a technical bottleneck that definitely requires a solution.